Detection of oxidative stress in tissue homogenate from krill exposed to oil

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Faculty of Science and Technology

MASTER’S THESIS

Study program/ Specialization:

Biological Chemistry

Autumn semester, 2014 Spring semester, 2015

Open access

Writer:

Linda Bærheim ………

(Writer’s signature)

Faculty supervisor: Kåre Bredeli Jørgensen External supervisor(s): Grete Jonsson Thesis title:

Detection of oxidative stress in tissue homogenate from krill exposed to oil

Credits (ECTS): 60sp

Key words:

krill, homogenization, oxidative stress, biomarkers, MDA, malondialdehyde, AOPP, advanced oxidative protein products, oil exposure, produced water, spectrophotometry, HPLC-F, fluorescence

Pages: 57

+ enclosure: 11

Stavanger, …14.07.2015…………..

Date/year

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Detection of oxidative stress in tissue homogenate from krill exposed to oil

Master’s thesis in Biological Chemistry

By

Linda Bærheim Spring 2015

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Acknowledgement

This thesis is performed in cooperation between University of Stavanger (UiS), International Research Institute of Stavanger (IRIS) and Stavanger University Hospital (SUS).

First, gratefully I would like to acknowledge my external supervisor Grete Jonsson at SUS, for all her help and great support during this thesis!

I really appreciate the opportunity of writing my thesis at IRIS, and I would like to thank Renée Katrin Bechmann and the rest of the SeaSens team for how they welcomed me on the team. Thanks to all the helpful people at IRIS, who answered all the questions I had, and gave me valuable assistance along the way. I would especially like to thank Kjell Birger Øysæd for all his support on the lab, and Eivind Larssen for his help with the high performance liquid chromatography (HPLC) system.

I am also very grateful to my supervisor at UiS Kåre Jørgensen for all his good tips on structuring the thesis and helping with the writing.

Finally, I also would like to thank my family, friends and especially my fiancée Geir for all their support!

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Abstract

Krill are small crustacean animals, living all over the world. They are key organisms in the marine food web. The petroleum industry releases oil to the sea with the produced water and by accidental oil spill, and there is a concern about the effect the oil has on krill. Discharges of oil can lead to a situation of oxidative stress in marine organisms. Biomarkers such as malondialdehyde (MDA) and advanced oxidative protein products (AOPP) can be used to detect oxidative stress.

Existing methods for homogenization, total protein content, AOPP and MDA were modified and validated for use on krill homogenate. The methods were then utilized to analyze krill from a study performed at the International Research Institute of Stavanger (IRIS), where krill had been exposed to sublethal oil

concentrations for eight days. The experiments were performed in spring, autumn and winter to investigate potential seasonal variations in biomarker response.

The following results were obtained:

 The homogenization process includes two successive centrifugation steps in order to get a clear and stable krill homogenate.

 The krill homogenate had to be pre-diluted for the spectrophotometric methods to get an absorbance within the linear range of the calibrators (1:10 or 1:15 for total protein and 1:8.3 or 1:13.3 for AOPP).

 Instrumental limits of detection and quantification for AOPP were 2.44 µM and 7.38 µM respectively, and the instrumental limit of quantification for MDA was 0.28 μM.

 The within-run variations were 7.8% and 4.0-13% for plasma control and krill homogenate respectively for AOPP, and 19% for krill homogenate control for MDA.

 Between-run variation was 8.8% for plasma control for AOPP, and 19% and 10% for krill homogenate control and plasma respectively for MDA.

 AOPP and MDA levels were significantly higher in krill that were frozen directly after capture (T0) in spring and autumn compared to krill kept in the laboratory (T1).

 Seasonal differences were detected with a significantly lower T1 MDA level in the spring krill, a significantly higher T0 MDA in autumn krill, and a significantly lower T0 AOPP in winter compared to T1 or T0 respectively of the other seasons.

 Any effect of the oil exposure however was not observed with MDA or AOPP.

Three methods, AOPP, MDA and total protein, were successfully adjusted and validated for analysis of krill homogenate. Due to the good sensitivity of the methods, individual krill could be analyzed for both AOPP and MDA, and the concentrations normalized with respect total protein content. AOPP was a simple and fast method, and with higher precision than the MDA. An improved AOPP could be the method of choice for the future monitoring of oxidative stress level in krill. However, changes in AOPP or MDA levels due to oil treatment for eight days were not detectable due to a high natural variation of these biomarkers in the krill homogenate.

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Abbreviations

AGE Advanced Glycation End Product

ALE Advanced Lipoxidation End Products

AOPP Advanced Oxidative Protein Products

BCA Bicinchoninic Acid

BHT Butylated Hydroxytoluene

BSA Bovine Serum Albumin

CA Citric Acid

CV Coefficient of Variation

DNPH 2,4-dinitrophenylhydrazine

DW Dry Weight

HPLC High Performance Liquid Chromatography

HPLC-F High Performance Liquid Chromatography with Fluorescence detector IRIS International Research Institute of Stavanger

IUPAC The International Union of Pure and Applied Chemistry

MDA Malondialdehyde

NFR Norges forskningsråd

PAH Polycyclic Aromatic Hydrocarbons

PBS Phosphate Buffered Saline

PDA Photodiode array

PROOF/PROOFNY Long-term effects of discharges to sea from petroleum-related activities. Research programs under NFR (2002-2015).

PUFA Polyunsaturated Fatty Acid

ROS Reative Oxygen Species

SeaSens Seasonal variation in sensitivity of krill to oil

SOP Standard Operating Procedure

SUS Stavanger University Hospital

TBA Thiobarbituric Acid

TBARS Thiobarbituric Acid Reactive Substance

TEP Tetraethoxypropane

TProt Total Protein

UiS University of Stavanger

UV Ultraviolet

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1 Introduction

This thesis is part of the “SeaSens - Seasonal variation in sensitivity of krill to oil” project at International Research Institute of Stavanger (IRIS) funded by Norges forskningsråd (NFR). The SeaSens project will investigate krill response to oil in produced water, with a focus on seasonal variation.

1.1 Krill

Krill (euphausiids) are small shrimplike crustaceans (Figure 1-1), and in the same species class

(Malacostraca) as shrimps, craps, lobsters and woodlice [1]. There are about 85 krill species spread all over the world [2], Northern krill (Meganyctiphanes norvegica) is the most common species in the north, where it habitats a large area in the North Atlantic from 30° N to 80° N, and the western part of the Mediterranean Sea [3]. The northern spawning limit for M. norvegica is around the Arctic Circle and it cannot truly be regarded as an Arctic species [4]. Thysanoessa is a genus that can be found in the north;

where Arctic krill (Thysanoessa raschii) is found as far north as 80° N in the Arctic Ocean [2]. T. inermis and T. longicaudata are the most common species in the Barents Sea [5].

Figure 1-1 The Northern krill (Meganyctiphanes norvegica). Modified from [6]

The majority of krill species live 0-400 m under the sea surface, with a diurnal vertical migration where they approach the surface at night [2]. Most krill are herbivorous with a diet of phytoplankton and algae, but some krill eat zooplankton as well [2]. The M. norvegica are carnivorous and can even eat their own species [2]. The krill are a part of the lower food chain, and they are prey for larger animals such as fish, squid, whale, penguin and seal [7].

Krill have a transparent body where the digestive system is visible (Figure 1-2). The food is mechanically processed in the stomach before the enzymes in the migut gland (hepatopancreas) further break it down [8]. Krill contain some red pigments (the carotenoid astaxanthin and its esters), that are assumed to

provide UV protection [9]. Their astaxanthin source is digested algae [9]. The krill live in the dark, and the Euphausiidae family all possess light organs (photophores) to produce blue light, where the 10 separate light organs of M. norvegica allow it to bioluminescence spontaneously via a luciferin-luciferase-type of biochemical reaction [10].

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Figure 1-2 The digestive system of M. norvegica in the cephalothorax area [8].

The lipid composition in krill varies with season and food sources available. Falk-Petersen et al. [4] found a total lipid content (% of dry weight DW, Figure 1-3) in M. norvegica that was highest in the winter months, suggesting a more herbivorous diet during the summer. The cephalothorax contains the

hepatopancreas that has the highest lipid content of all the krill organs (65% DW). The cephalothorax also contains the stomach (17% DW lipid content), the gonads (34% DW lipid content) and fat body of

connective tissue (20% DW lipid content) [8]. The abdomen contains mostly muscle tissue with only 8%

(DW) of lipids [8]. A high lipid store may leave the animals more disposed for oxidative stress, as lipids are preferred targets of reactive oxygen species (ROS) [11].

Figure 1-3 Lipid dynamics (total lipid) of M. norvegica in relation to season. The spring bloom period of phytoplankton is indicated. Modified from [4].

1.2 Produced Water

Krill from the SeaSens project were exposed to oil concentrations that represent discharges of produced water. Produced water is water brought to the surface with the crude oil or the natural gas. Either it was a natural part of the oil and gas in the reservoir, or water injected to the reservoir to increase the extraction of oil and gas. When the oil or gas is processed, this water is separated out, and the produced water must

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be treated according to government regulations to remove oil and grease before the water is released to the environment [12].

The produced water contains naturally-occurring compounds such as inorganic salt, metals and metalloids, and a large variety of chemicals used in the water treatment offshore such as emulsifiers, surfactants, oil removing agents and scale inhibitors [12]. Typical organic compounds in the produced water include aliphatic hydrocarbons, carboxylic acids, phenols and low molecular weight aromatics, and polycyclic aromatic hydrocarbons (PAH). There is a maximum limit of 30 ppm (mg/L) oil in water in Norway [13].

The existing toxicity of PAH on different organisms can be drastically enhanced when the oil is exposed to sunlight, either by photosensitivity and production of singlet oxygen, or modification to a more toxic compound [14]. The photoinduced toxicity has been demonstrated in PAH like anthracene, fluoranthene and pyrene [15]. The toxicity of PAH increases from 2 to over 1000 times in the presence of UV light [16].

The long-term effects of discharges to sea from petroleum-related activities have been investigated in research programs PROOF and continued PROOFNY for over ten years [13]. The programs have found negative effects on different areas like bile metabolites in cod, sex hormone-mimicking effect on rainbow trout and egg development in mussels. Bechmann et al. [17] investigated Northern shrimp (Pandalus borealis) exposed to oil in water (0.015, 0.06 and 0.25 mg/L) and found that the amount (concentration and time) of the exposure correlated to PAH accumulation in tissue. The amount of the exposure also correlated to the biomarker responses; the lysosomal membrane stability (a general health indicator) and the alkaline unwinding (indication of DNA damage). When PAH is taken up by an organism, it can stimulate the production of reactive oxygen species (ROS) and lead to oxidative stress. This has been demonstrated in the Artic scallop (Chlamys islandica) with increasing lipid peroxidation over time when exposed to oil [18].

1.3 Krill Exposure Study

The SeaSens krill project is a part of the PROOFNY program, with a particular interest in studying Arctic and temperate species. To their knowledge at the time of application, no other studies had investigated the seasonal variation in response to oil for any species in the North Atlantic or Barents Sea. The krill are an important link in the marine food web [10] and are key organisms in temperate and Arctic ecosystems.

The link between the phytoplankton, the krill and their predators such as the blue whale is potentially one of the shortest food chains involving a large marine mammal [19]. Krill are highly nutritious with their vitamin, mineral, essential amino acid and ω-3 polyunsaturated acid levels [20]. The effect of the oil exposure in different seasons could potentially vary as the physiological composition of the krill varies with the seasonal variation in type of food and the availability of the food.

The exposure study was conducted on the krill species M. norvegica, as a representative for all krill species in the Barent Sea. The oil concentrations the krill were exposed to reflect produced water

discharges in low (0.015 ppm) and high (0.15 ppm) oil concentrations. In spring and summer seasons the oil might be photomodified due to UV radiation, potentially increasing the toxicity of the oil. The SeaSens project (Figure 1-4) will analyze several parameters in the krill that might be affected by the oil exposure.

They are looking at parameters such as histology of gills and digestive glands, fatty acid composition, feeding rate, gene expression, oxidative stress and polycyclic aromatic hydrocarbons (PAH) in krill tissue.

These parameters will be used in the investigation if krill has a seasonal variation in how it is affected, and

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if the ultraviolet (UV) irradiation of oil in the water will increase the toxicity of oil. The relevant

biomarkers will then be established as methods for monitoring the effects produced water discharges has in the field.

Figure 1-4 The SeaSens krill experiment. The krill captured in each season are exposed to different oil treatments.

After the exposure experiment the krill will be analyzed for several parameters, including oxidative stress. (Figure from the SeaSens project description, reused with permission.)

1.4 Purpose of Thesis

Oxidative stress is an important component of the stress response in marine organisms exposed to pollution such as oil discharges from the petroleum industry [21]. By measuring the biomarkers of oxidative stress it can be possible to monitor the effects produced water has on krill in the field. The goal of this thesis is to establish a method for detecting oxidative stress in krill homogenate, by considering the biomarkers advanced oxidative protein products (AOPP) and malondialdehyde (MDA). MDA is a

relatively stable end product of lipid peroxidation, and AOPP is a measurement of protein oxidation. The thesis used existing methods for measuring MDA and AOPP, and optimized them for use on krill

homogenate. AOPP has never been measured in krill before. These methods should be easily implemented at the IRIS facilities by using the existing equipment. The aim is to find a method that can be used to monitor the effects of oil exposure on krill in the field.

The thesis involved multiple steps to achieve the end results of protein normalized AOPP and MDA values (Figure 1-5). The homogenate was prepared from the krill thorax, as the whole krill was used for both the genomics and oxidative stress analyses to be able to increase the number of samples. The aim was to prepare and store the homogenate under such conditions that total protein, AOPP and MDA could be analyzed with minor influence of pre-analytical factors. The homogenization process disrupt the cell wall, and is usually done at low temperatures (1 – 4 °C) to minimize the activity of krill endogenous proteases [22]. The homogenization could either be performed by a common, basic procedure using a grinder, or more extensive processes with liquid nitrogen. The centrifugation of the ground tissue separates the

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different components of the tissue cells, leaving the insoluble parts such as shell, appendages and

insoluble proteins in the pellet [22]. The existing methods for homogenization [23], total protein [24, 25], AOPP [26] and MDA (unpublished method from my supervisor) were modified and validated. It was important to find the optimal pre-dilutions of the homogenates to get absorbance within the calibration standards for the AOPP and the total protein methods. For the MDA method, the calibration standard concentrations were increased from the original method to reflect the MDA in the krill homogenates.

Figure 1-5 Overview of the processing of the krill. Details of the methods in chapter 3.

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2 Theory

This chapter gives background information about oxidative stress and biomarkers for oxidative stress, and presents some measurement principles that will be utilized in this thesis.

2.1 Oxidative Stress

Oxygen is vital in biochemical processes of aerobic organisms, and reactive oxygen species (ROS) have a natural part in the cellular metabolism and functions as a defense against pathogens. The main source of ROS is leakage from the mitochondrial electron transport chain [27, 28]. ROS is oxygen in a more

reactive state than molecular oxygen (Table 2-1). AOPP formation has been linked to chlorinated oxidants such as hypochlorous acid or chloramines [29] and not to 𝑁𝑂₂^∙ [30]. Transition metals like iron (Fe²⁺/Fe³⁺) and copper (Cu⁺/Cu²⁺) are remarkably good promoters of free radical reactions, and they can convert O2·-

and H2O2 into the highly reactive ^·OH [31].

Through evolution the cells have developed several antioxidant defense mechanisms to protect it from ROS [32]. Antioxidants can significantly delay or prevent oxidation of a molecule [33] and can be divided into enzymatic and nonenzymatic antioxidants. The enzymatic antioxidants includes the superoxide dismutase which is a catalyst of 𝑂₂^∙−, the catalase which is a catalyst of H2O2 and the glutathione

peroxidase working together with glutathione which is highly abundant in animal tissues and catalyze the reduction of H2O2 [21]. The nonenzymatic includes glutathione, ascorbic acid (vitamin C) which is a reductant source for H2O2, 𝑂₂^∙−, 𝑂𝐻^∙ and lipid hydroperoxides, and carotenoids, where the latter can either function as a light-harvesting pigment or quench ROS produced from an overexcitation by light [21].

Astaxanthin, a carotenoid found in krill, is a powerful antioxidant [34].

Table 2-1 List of some reactive oxygen species (ROS). Modified from [35].

Free radicals Nonradicals

Superoxide, 𝑂₂^∙− Hydrogen peroxide, 𝐻2𝑂2

Hydroxyl, 𝑂𝐻^∙ Hypobromous acid, 𝐻𝑂𝐵𝑟

Hydroperoxyl, 𝑂𝐻₂^∙ Hypochlorous acid, 𝐻𝑂𝐶𝑙

Peroxyl, 𝑅𝑂₂^∙ Ozone, 𝑂₃

Alkoxyl, 𝑅𝑂^∙ Single oxygen, (𝑂₂¹∆𝑔) Carbonate, 𝐶𝑂₃^∙− Organic peroxides, 𝑅𝑂𝑂𝐻 Carbon dioxide, 𝐶𝑂₂^∙− Peroxynitrite, 𝑂𝑁𝑂𝑂⁻ Nitric oxide, 𝑁𝑂^∙ Peroxynitrous acid, 𝑂𝑁𝑂𝑂𝐻 Nitrogen dioxide, 𝑁𝑂₂^∙ Chloramines

Oxidative stress (Figure 2-1) is a situation that occurs when there is a serious imbalance between the antioxidants and the reactive oxygen species (ROS) that can damage the cell [35]. The imbalance means there are more of ROS than there normally should be. This could be due to an increase of ROS, or a decrease of antioxidants. The cell will be less capable of defending itself, and there could be extensive damage to nucleic acids, lipids and proteins [21]. Proteins are the major target for ROS (50 - 75% of all ROS), and all from primary to quaternary structure may be changed by mechanisms such as peptide backbone cleavage, cross-linking and/or modification of the side-chain of virtually every amino acid [36].

The pathogenesis or the progression of most human diseases such as cancer, cardiovascular and

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neurodegenerating diseases, and even the aging process, have been linked with increased oxidative damage [37].

Figure 2-1 Imbalance between oxidants and antioxidants leads to oxidative stress.

2.2 Biomarkers for Oxidative Stress

A commonly used definition of a biomarker is “a characteristics that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic intervention” [38]. It could be biochemical, physiological, histological,

morphological or behavioral measurements, and the biomarker could either be to indicate exposure of a chemical (without information on degree or effect), or to indicate effect of expose (the toxic effect on the organism) [39].

It is difficult to use the ROS as biomarkers for oxidative stress, as they are not stable long enough to be measurable. The fingerprint left by the ROS could be measured however. ROS leaves stable metabolites and oxidation target products like lipid peroxide end products and oxidized proteins [36].

Malondialdehyde (MDA) is the most commonly used biomarker for lipid peroxidation [32], and the most commonly utilized way of measuring protein oxidation is by detection of carbonyl groups [40] (Table 2-2).

Table 2-2 The most common biomarkers for the different types of oxidation. Derived from [32].

Type of oxidation Biomarker Method

Lipid Malondialdehyde

(MDA)

Derivatization with thiobarbituric acid (TBA), and detected by fluorometry, spectrophotometry, HPLC- VIS or HPLC-F^*

DNA Guanine hydroxylation (8-OHdG)

Detected by an electrochemical detector (ECD) connected with HPLC, or immunoassay with spectrophotometry

Protein Protein carbonyl Derivatization with 2,4-dinitrophenylhydrazine (DNPH), and detected by spectrophotometric assay, enzyme-linked immunosorbent assay (ELISA) electrophoresis followed by Western blot immunoassay

*high performance liquid chromatography (HPLC), visual light (VIS), fluorescence (F).

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2.2.1 Malondialdehyde (MDA)

Malondialdehyde (MDA) is a small (72.07 g/mol), volatile, three carbon dialdehyde with pKa of 4.46 for the enolic OH group [41]. In a neutral or alkaline environment it will mainly be in its enol form (Figure 2-2) [42]. With pH at physiological conditions the MDA molecule is moderately reactive, and is considered highly toxic due to its potentially mutagenic and atherogenic interaction with DNA and proteins [42]. MDA is formed by lipid peroxidation (Figure 2-3), and is the most studied product of polyunsaturated fatty acid peroxidation [43].

Figure 2-2 The MDA molecule in its keto and enol form. IUPAC name: propanedial.

Figure 2-3 Lipid peroxidation. LH is the polyunsaturated lipid, L^∙ is the carbon-centered lipid radical, LOO^∙ is the lipid peroxyl radical and LOOH is the lipid hydroperoxide. Termination of the lipid peroxidation happens when lipid radicals interacts and/or forms nonradical species [32]. The LOOH can easily decompose into lipid alkoxyl radicals (𝐿𝑂^∙), aldehydes, alkanes, lipid epoxides and alcohols [32]. Modified from [44].

The MDA molecule can exist in free form or bound to matrix molecules [42]. It is usually measured after derivatisation with thiobarbituric acid (TBA) which reacts with the free MDA under acidic conditions to create a MDA(TBA)2 derivate (Figure 2-4), first described by Yagi (1976) [45]. The acid will hydrolyze some of the bound molecules to allow them to react with the TBA. A similar reaction is achieved with base hydrolysis. The MDA(TBA)2 derivate has a red chromophore that can be detected

spectrophotometrically at 532 nm [46], or fluorometrically with excitation/emission (ex/em) at 525/560 nm [47] or 515/553 nm [48]. The MDA molecule itself has no eletrophore, chromophore or fluorophore [43]. 1,1,3,3-Tetraethoxypropane or 1,1,3,3-tetramethoxypropane that quantitatively converts to MDA

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when heated in acidic solution can be used as calibration standards [45, 48], and derivatized in the same manner as the samples. There are several different compounds that can react with TBA (TBA-reacting substances, TBARS), not just MDA. The other TBARS could be other aldehydes, sugars, amino acids, bilirubin and albumin [43], which will lower the specificity of the method, and is one of the reasons why the method has been criticized over the years.

Figure 2-4 MDA reaction with TBA[43]. Two TBA molecules reacts with the MDA molecule to form the MDA(TBA)2

chromogen under acidic conditions.

To improve the method specificity a chromatography step has been introduced. High pressure liquid chromatography (HPLC) or gas chromatography (GC) will separate some of the other chromogens from the MDA(TBA)2 complex [41, 49]. The fluorescence detector (section 2.4) also improves the specificity compared to spectrophotometry by only detecting the chromophores with fluorescent abilities. The highest selectivity/specificity is achieved with mass spectrometry methods like GC-MS/MS or LC-MS/MS [37].

MDA in human plasma samples was reported stable for at least 6 months when stored at -80 °C [50]. The amount of MDA in a sample may vary due to how the sample is treated and stored. The derivatization step includes a hydrolysis step releasing the bound MDA. Acidic hydrolysis has been the most commonly used, but alkaline hydrolysis gives higher reported MDA [51]. This is either a more efficient way to free the bound MDA or it could be MDA formed from hydroperoxides in addition to the protein-bound MDA [52]. The lipid peroxidation could continue in vitro, triggered by the low pH and high temperature, which will give a higher MDA level; or the MDA molecule could be oxidized to a carboxylic acid or reduced to an alcohol, lowering the detectable MDA. Treating the samples with an antioxidant like butylated hydroxytoluene (BHT) could lower the artefactual lipid peroxidation [53]. However, the effect of BHT is under debate as some have not found any difference when not including the BHT in the derivatization process, and others found that the effect did not come from BHT but from the ethanol (or methanol) it was dissolved in [43].

The reported MDA level in human plasma varies depending on the method used. Del Rio et al. [42]

presents a selection on MDA findings, where the level varies from 0.11 ± 0.03 μmol/L (TBA with HPLC- UV/Vis or with fluorimetry), 0.69 ± 0.13 μmol/L (TBA with HPLC-F) up to 13.8 ± 1.32 μmol/L (2,4- dinitrophenylhydrazine (DNPH) derivatization with HPLC-UV/Vis). TBARS levels in three different krill species have been reported as about 0.25 μmol/g protein in cold season (January), and 0.002 – 15 μmol/g protein in warm season (July and October) [54].

2.2.2 Advanced Oxidation Protein Products (AOPP)

The term ‘advanced oxidation protein products’ or AOPP was introduced by Witko-Sarsat et al. [55] in 1996 as novel oxidative stress marker of oxidized proteins. The plasma of uremic patients and healthy control was measured spectrophotometrically (section 2.3), and a peak at 340 nm was found for the uremic patients. This indication of a new chromogen correlated with plasma levels of the protein oxidation biomarkers dityrosine and pentosidine, but not with TBARS as the biomarker for lipid peroxidation. There is not necessarily correlation between biomarkers of protein oxidation and biomarkers of lipid

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peroxidation, as there are different repair mechanisms and different degradation in vivo [35]. AOPP was further investigated in 2004 by Capeillère-Blandin et al. [30], and two overlapping chromophoric bands with maxima at 310 nm and 340 nm were found, corresponding to dityrosine, carbonyl proteins and pentosidine.

Dityrosine is a specific marker for free radical modification and oxidation of proteins, since it can only be formed by a reaction of two tyrosyl radicals [56]. The dityrosine is among other mechanisms synthesized by the myeloperoxidase-H2O2 system [57]. Dityrosine is metabolically stable, and increased

concentrations have been found in various conditions as atherosclerosis, cystic fibrosis, end-stage renal disease and acute inflammation with or without sepsis [36]. Carbonyl group generation on proteins can be a result of:

1) The oxidation of amino acids, especially proline, arginine, lysine and threonine [40].

2) Advanced lipoxidation end-products (ALE) which are Michael adducts formed between lysine, histidine or cysteine and α,β-unsaturated aldehydes [36].

3) Advanced glycation end products (AGE) which are glycation/glycoxidation of lysine [36].

Pentosidine is a fluorescent AGE structure, which involves lysine and arginine residues combined with an imidazo-(4,5b)-pyridinium ring [58]. Pentosidine is considered a specific biomarker of protein glycation [29].

The AOPP levels in plasma were found stable for at least 6 months when stored at -80 °C [59], but protein carbonyls have been demonstrated as stable for 10 years when stored at -80 °C [36]. The original AOPP method was based on a dilution of plasma in phosphate buffered saline (PBS) and acetic acid, and

calibration standards based on potassium iodide (KI) oxidized by chloramine-T (Figure 2-5) [55, 60]. The AOPP concentrations are expressed as μM chloramine-T equivalents, but commonly abbreviated to μM.

Chloramine-T is a stable and strong oxidant and electrolyte in both acidic and alkaline media with Ered=1.138 at pH 0.65 and Ered=0.5 at pH 12, and with pKa estimated to 9.5 [61]. Chloramine-T can oxidize the iodide to iodine [61], which together with iodide forms the water-soluble triiodide (𝐼₃⁻) (eq 1 and eq 2). The triiodide can be measured spectrophotometrically, where it has absorption peaks at 290 nm and 351 nm [62].

Figure 2-5 Two structures of chloramine-T. Structure ‘A’ to the left is the most commonly depicted. Figure redrawn from Campbell and Johnson. [61].

2 𝐼⁻(𝑎𝑞) 𝐶ℎ𝑙𝑜𝑟𝑎𝑚𝑖𝑛𝑒−𝑇

→ 𝐼₂(𝑠) (1)

𝐼₂(𝑠) + 𝐼⁻(𝑎𝑞) 𝐼₃⁻(𝑎𝑞) (2)

Hanasand et al. [26] modified the AOPP method by improving the solubility of lipoproteins. This was achieved by using citric acid (0.2 M) to dilute the plasma samples. Soluble lipoproteins prevents overestimation due to light scattering, and removing the lipoproteins with a protein precipitation step

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would have caused an underestimation of the AOPP levels. The AOPP detected with this method was 82.6 ± 1.1 μM in medium AOPP plasma control.

There has been some confusion on how to use the method of Witko-Sarsat et al. [55]. In many cases the KI was added to the samples as well as to the chloramine-T [63-66], increasing the measured AOPP. This means that the reported AOPP results by others must be considered with this in mind, even for those who do not describe in detail how they prepared their samples (and thus reveal that KI was added to the sample). Altan et al. [67], Inkielewicz-Stępniak and Knap [68] and Benedetti et al. [69] all investigated AOPP levels in rat liver tissue homogenates, but reported 1.72 ± 0.32 nmol/g protein, about 110 μmol/g protein and about 15 μmol/g protein respectively in their healthy control samples, differing by a factor up to 100.

2.3 Spectrophotometry

Spectrophotometry is about measuring how light interacts with matter, it can be transmitted right through a solution, it can be reflected, scattered, absorbed and re-emitted [70]. Molecules that are capable of absorbing light are called chromophores, they excite to a higher energy state for a brief moment before returning to the ground state [71]. The energy is absorbed in double and triple bonds of unsaturated organic compounds [72]. The absorbance in a solution is defined as the logarithmic difference in radiant power of the incident beam and the beam that has passed through. This attenuation is dependent of how many chromophores it passed, as defined by the concentration of the solution and the path length, and is known as Beer-Lamberts law [72]:

A = log (P0/P) = εbc (3)

where P0 and P are the radiant power of incident beam and beam transmitted through respectively, ε is a proportionality constant for a given wavelength, b is the path length of the medium and c is the

concentration of the absorbing species. The law holds as long as there is no interaction between solution molecules, and the concentration is not too high [73].

A microplate spectrophotometer (Figure 2-6) measures the intensity of light as a function of wavelength in a 96 well plate. This well plate includes calibration standard solutions with known concentrations of substance that absorbs at the given wavelength in addition to samples with unknown concentrations. The samples with unknown concentrations must have absorption within the range of the calibration standards.

If not, the samples must be diluted, or the calibration standard concentrations adjusted. With the same path length (volume of the samples) and intensity of the incident light in all wells, the unknown concentrations of analyte in the samples can be determined using the line of regression. The spectrophotometry method has low specificity though, as multiple substances in the sample may absorb at the given wavelength [74].

In addition, particles or precipitation in the sample will increase the measured absorbance by scattering or reflection of the incident beam.

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Figure 2-6 The Multiskan Ascent plate reader (Thermo LabSystems) at IRIS. It is capable of measuring both in the ultraviolet (UV) area (340 nm) and in the visible (VIS) light range (595 nm).

2.4 High Performance Liquid Chromatography – Fluorescence

Chromatography is a collective term for separation techniques based on distribution between two phases.

The substances in the sample are transported by a mobile phase (gas or liquid) through the stationary phase where the substances adsorbs back and forward between the two phases. Separation occurs as the substances have different chemical qualities that affect the affinity to the different phases and thus the time spent in the stationary phase.

Figure 2-7 HPLC system - schematic setup. The minimum of elements included in a HPLC system. Derived from [75].

In a High Performance Liquid Chromatography (HPLC) system the solvent (liquid mobile phase) is pumped from a reservoir at a given flowrate, and the autosampler injects a small portion of the sample to the flow that is sent through the column with packed material (stationary phase) where the separation occurs under pressure (Figure 2-7). The separation could be done in several chromatographic modes based on the different properties of the molecule; by polarity, charge, size or ligand specificity. Separation by polarity with the reversed-phase chromatography (Figure 2-8) is the most commonly used [76], and is the method that was used for separation of the MDA derivate in this thesis. With reversed-phase

chromatography, the polar mobile phase will be water mixed with a polar organic solvent like acetonitrile or methanol, and the non-polar column can have hydrophobic alkyl chains bound to very small porous particles [72]. The non-polar molecules in the mobile phase will tend to adsorb to the surface on the inside and outside of a particle if that surface is also non-polar. This means that the polar molecules will spend the least time in the stationary phase and will be eluted first. Elution of the non-polar molecules is accomplished by increasing the polar content of the mobile phase.

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Figure 2-8 Reversed-phase chromatography principle. The non-polar molecules (green) will bind to the non-polar hydrocarbon tails of the stationary phase (typically C8 or C18 tails on silica bead), while the polar molecules (blue) will not bind.

After separation, the components of the sample elute from the column and are sensed by a detector. The detected signal forms a chromatogram with signal strength versus time. The components should ideally have been completely separated by this step, for each to have its own peak in the chromatogram. The signal should be higher than the baseline noise for a positive identification (Figure 2-9), with a signal-to- noise (S/N) above 3. The time to elute is called the retention time (tRT), from once the sample was injected to the center of the peak, and should be at some distance from retention time t0 of the non-retained

compounds. The substances are normally identified based on the order they emerge from the column and by their retention time. The retention time can be adjusted by changing parameters such as type of mobile phase and flow, column, temperature and injection sample size. The reversed-phase column used in this thesis was 75 mm long with internal diameter of 2 mm, and had C18-coated particles of size 3 μm. The HPLC method had a gradient elution with phosphate buffer and methanol, and by varying the combination of the aquatic solvent (A) and organic solvent (B) over time the different substances were eluted.

Figure 2-9 The signal (S), noise (N) and retention time (tRT) of the MDA(TBA)2 derivate. The chromatogram (for the lowest concentration calibration standard) also show the retention time t0 for the nonretained compounds.

Fluorescence

In this thesis a fluorescence detector was used in combination with the HPLC (HPLC-F) to detect the chromophore of the MDA(TBA)2 derivate.

A fluorescence detector is using the fluorescence abilities in the analyte, where energy in photons with a given wavelength can be absorbed, and then reemitted at a longer wavelength with some loss of energy (heat loss) (Figure 2-10). The exact wavelengths the fluorophore is excited at and emitted at is molecule specific. Unlike the UV/VIS detectors that measures in the direction of the incident light, a fluorescence sensor is usually at a 90° angle, to minimize the risk of detecting transmitted or reflected incident light.

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The SpectraSYSTEM FL3000 fluorescence detector used in this thesis has monochromators for the excitation and emission wavelengths. The unique set of excitation and emission means that a fluorescence detector is more specific than a UV/VIS detector.

Figure 2-10 Jablonski energy diagram fluorescence [77]. The incident light is absorbed, and then energy is emitted in all directions at a different wavelength.

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3 Materials and Methods

3.1 Chemicals and Reagents

Bradford Reagent, potassium dihydrogen phosphate (KH2PO4, anhydrous, ≥99.7%), chloramine-T (CH3C6H4SO2NClNa·3H2O, ≥99%), potassium iodide (KI, ≥99.5%), 2,6-bis(1,1-dimethylethyl)-4- methylphenol (BHT, ≥99.0%), 2-thiobarbituric acid (TBA, ≥98%) and 1,1,3,3-tetraethoxypropane (TEP,

≥96%) were purchased from Sigma-Aldrich (Steinheim, Germany). Potassium hydrogen phosphate (K2HPO4, p.a.), sodium chloride (NaCl, ≥99.5%), citric acid monohydrate (C6H8O7 · H2O, ≥99.5%), glacial acetic acid (CH3COOH, anhydrous, ≥99.8%), sulfuric acid (H2SO4, 95-97%, p.a.), 1-butanol (CH3(CH2)3OH, p.a.), potassium hydroxide (KOH, ≥85.0%), methanol (CH3OH, gradient grade for liquid chromatography, ≥99.9%) were purchased from Merck (Darmstadt, Germany). Phosphate buffered saline (BupH^TM, 0.1 mol/L sodium phosphate, 0.15 mol/L sodium chloride, pH 7.2) was purchased from Thermo Scientific (Rockford, USA). Bovine serum albumin (BSA, Albumin fraction V fatty acid free) was

purchased from Roche (Mannheim, Germany). Spinal Fluid Control (Liquichek, level 2) was purchased from Bio-Rad Laboratories (Irvine, USA). Absolute ethanol (prima) was purchased from Kemetyl (Vestby, Norway), and phosphotungstic acid (H3O40PW12.xH2O) was purchased from VWR (Leuven, Belgium).

3.2 Equipment

Table 3-1 List of equipment with details on model and manufacturer.

Equipment Model Manufacturer

Overhead stirrer for homogenization. Used with VWR homogenization grinder (10 mL) and plain plunger/piston

Eurostar, 50-2000 rpm IKA. Janke & Kunkel GMBH & Co

Centrifuge 5415 R Eppendorf

Vacuum centrifuge Concentrator 5301 Eppendorf

Microplate reader.

Used with

- PS Microplate 96 well flat bottom (Greiner Bio-One) for Total Protein

- Costar UV Plate, 96 well, with UV transparent flat bottom (Corning Incorporated, VWR Norge) for AOPP

Multiskan Ascent Thermo LabSystems

HPLC-F system processed with Empower software:

-LC column Gemini NX 3u C18 110A Phenomenex

-PDA detector WatersTM 996

Photodiode Array Detector

Waters

-Fluorescence detector SpectraSYSTEM FL3000 Thermo Separation

Products

-Autosampler WatersTM 717plus

Autosampler

Waters

-Controller WatersTM 600 Controller Waters

-Degasser DEGASYS DG-2410 Uniflows

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3.3 Solutions

Buffer A

Buffer A (0.1 M phosphate) for homogenization was prepared according to IRIS Standard Operating Procedure (SOP) ‘Preparation of S12, microsomes and S100 by differential centrifugation’ [23]. 3.4 g K2HPO4 (0.1 M) was dissolved to 0.5 L with distilled water, and 8.75 g KH2PO4 (0.1 M) was dissolved to 0.25 L with distilled water. Some of the KH2PO4 solution was added to the K2HPO4 solution until a pH of 7.4 was achieved in the mixture. The mixture was added NaCl (12.5 g pr. 0.5 L of mixture). The buffer was kept at +4 °C (stable for 6 months [23]).

PBS Buffer

The phosphate buffered saline (PBS) buffer (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2) was made by dissolving one pack of BupH^TM phosphate buffered saline to a total volume of 500 mL with distilled water. The buffer was kept at +4 °C.

Total Protein Calibration Standards

Bovine serum albumin (BSA) was weight (1 g) and dissolved in 10 ml distilled water as a stock solution.

Since BSA is hygroscopic, the accurate concentration was determined spectrophotometrically by diluting the stock solution 1:100 in PBS buffer (50 μL + 4950 μL = 1 g/L). Absorbance for 1 g/L at 280 nm should be 0.667 [78]. This was measured to 0.635, thus the BSA stock was 95.2 g/L. Calibration standard

solutions (Table 3-2) were made from stock BSA diluted in PBS buffer. The calibration standards were made with five different concentrations between 0.143 mg/mL and 1.24 mg/mL. The stock BSA was stored in 500 μL aliquots at -20 °C, while the calibration standard solutions were kept at +4 °C (no longer than 3 months).

Table 3-2 The calibration standards for Total Protein. The BSA stock solution had a protein content of 95.2 mg/mL.

Standard

Concentration BSA stock PBS buffer

(mg/mL) (μL) (μL)

2 0,143 7,5 4992,5

3 0,286 15 4985

4 0,571 30 4970

5 0,857 45 4955

6 1,24 65 4935

Citric Acid

Three different citric acid (CA) concentrations (0.1 M, 0.2 M and 0.5 M) were prepared in distilled water, and kept at +4 °C until use.

KI

Potassium iodide solution (KI, 1.16 M in PBS buffer) was prepared fresh each day, and protected from light exposure until use.

Chloramine-T Calibrator Standards

Chloramine-T stock solution (1 mM) was prepared in distilled water, and kept at +4 °C until use (no longer than 3 months). Seven calibration standards (Table 3-3) were prepared fresh each day from stock chloramine-T diluted in citric acid (0.2 M), with concentrations between 5 μM and 100 μM. When the

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calibration standards were added to the microplates they were further diluted by the addition of KI, thus a different concentration were used to create the standard curve (Table 3-3).

Table 3-3 The AOPP chloramine-T calibration standards. The calibration standards were prepared from stock (1 mM) and citric acid (0.2 M). 190 μL of calibration standard was added to the microplate, and when the KI (1.16 M, 10 μL) was added the actual chloramine-T concentration on plate changed accordingly. The ‘Chloramine-T on plate’

concentration was used to create the standard curve.

Standard Chloramine-T Chloramine-T CA (0.2 M) Chloramine-T

on plate (μM) stock (μL) (μL) (μM)

Cal 1 4,75 5 995 5

Cal 2 9,5 10 990 10

Cal 3 19 20 980 20

Cal 4 38 40 960 40

Cal 5 57 60 940 60

Cal 6 76 80 920 80

Cal 7 95 100 900 100

Butylated hydroxytoluene

The butylated hydroxytoluene solution (BHT, 0.7 mM in 40% ethanol) was prepared from a stock solution of 2,6-bis(1,1-dimethylethyl)-4-methylphenol (7.13 mM in absolute ethanol). The solution was kept at room temperature.

Thiobarbituric acid

2-Thiobarbituric acid (TBA) (46 mM in 50/50 glacial acetic acid/distilled water) was dissolved with magnetic stirring and gentle heating. The solution was kept at room temperature protected from light exposure.

Phosphotungtic acid

Phosphotungstic acid (2 g) was dissolved in 20 mL distilled water (10% w/v). The solution was kept at +4

°C.

Tetraethoxypropane

All solutions were prepared daily. The 1,1,3,3-tetraethoxypropane (TEP) was used to prepare several stock solutions in 40% ethanol (Table 3-4). With a TEP weight of 10.21 mg for approximately 10 μL, stock 1 has a concentration of 29.7 mM. Eight calibration standards were prepared from stock 3 diluted in 40%

ethanol (Table 3-5), with a concentrations from about 0.29 μM to 22.8 μM, in addition to blank (40%

ethanol).

Table 3-4 Stock solutions for TEP. Stock 1, 2 and 3 were prepared from TEP and the previously made stock solutions.

Stock 40% ethanol (μL)

TEP

(μL) Stock 1

(μL) Stock 2 (μL)

1 1490 10

2 990 10

3 900 100

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Table 3-5 The MDA calibration standards. Stock 3 has a TEP concentration of approximately 29 μM in 40% ethanol.

ID Stock 3

(μL)

40% ethanol (μL)

Concentration (μM)^a

Cal 2^b 10 990 0,29

Cal 3 20 980 0,57

Cal 4 40 960 1,14

Cal 5 80 920 2,28

Cal 6 160 840 4,56

Cal 7 320 680 9,13

Cal 8 500 500 14,3

Cal 9 800 200 22,8

a The concentration changes with the exact weight of the stock solution, example concentration with 9.80 mg TEP to stock.

b The standard wasnot used when analyzing the first four MDA analysis of krill exposed to oil (18 analyses in total).

Phosphate buffer for MDA

KH2PO4 (50 mM, distilled water) was adjusted to pH 7.0 with KOH (6 M, distilled water). The buffer was filtered through a membrane filter (pore size 0.45 μm) and kept at +4 °C.

Mobile phase ‘A’ for MDA HPLC

Mobile phase ‘A’ was 10% methanol and 90% phosphate buffer for MDA. The solution was kept at room temperature.

Mobile phase ‘B’ for MDA HPLC

Mobile phase ‘B’ was 90% methanol in distilled water. The solution was kept at room temperature.

3.4 Krill

The M. norvegica krill caught at night in the fjords outside of IRIS’ locations in Mekjarvik was used in the experiment. The krill were part of the SeaSens experiment (Figure 1-4, Table 3-6), but the summer setup was excluded from the SeaSens experiment due to poor krill harvest. An additional test was included in the autumn where krill were exposed to a higher oil concentration (1 mg/L of produced oil) for two days.

The T0 krill were frozen in liquid nitrogen right after capture and kept in a -80 °C freezer in cryovials. The T1 krill were acclimatized for about a week in the laboratory, prior to the eight days of exposure study. T1 krill were kept in separate containers, fed with EZ larvae and Artemia, and frozen in liquid nitrogen once the exposure was over.

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Table 3-6 The different krill groups. In total 75 krill were dedicated to each T1 treatment (Control, Low, High and Low UV) per season, where 15 krill of each treatment were allocated to oxidative stress analysis. The additional High High treatment consisted of 16 krill to oxidative stress analysis.

Season Group Use

Spring T0 method Frozen after capture. Used to develop the methods.

(captured 11.03.14)

T0 control Frozen after capture.

T1 experiment Frozen after eight days of exposure. Four treatments; Control (no oil exposure), Low (0.015 mg/L oil in seawater), High (0.15 mg/L oil in seawater) and Low UV (0.015 mg/L oil in seawater where the oil is treated with UV light).

Autumn T0 method Frozen after capture. Used to develop the methods.

(captured 23.09.14)

T0 control Frozen after capture.

T1 experiment Frozen after eight days of exposure. Three treatments; Control (no oil exposure), Low (0.015 mg/L oil in seawater) and High (0.15 mg/L oil in seawater).

Additional High High experiment of two days of exposure (1 mg/L oil in seawater).

Winter T0 control Frozen after capture.

(captured 14.01.15)

T1 experiment Frozen after eight days of exposure. Three treatments; Control (no oil exposure), Low (0.015 mg/L oil in seawater) and High (0.15 mg/L oil in seawater).

3.5 Homogenization

Homogenization was performed according to IRIS Standard Operating Procedure (SOP) [23] with some small modifications, using buffer A. Once removed from the -80 °C freezer, the krill were kept on ice (maximum four krill at once to avoid thawing quickly). The head and abdomen were cut off to get the thorax containing the hepatopancreas (Figure 1-2, Figure 3-1). The krill used during method development and the krill from the exposure study were treated slightly different. During method development the krill were kept on ice, then the thorax was separated and further homogenized. The krill from the exposure study had their head and abdomen separated from the thorax in a separate job where the krill were kept frozen on dry ice the whole time, as the head and abdomen were included in the separate genomics test.

The homogenization was done on thorax segments from the -80 C freezer (maximum four out at once, kept on ice).

Figure 3-1 Cut sections on krill. The thorax was separated from head and abdomen while still frozen.

The thorax was weighed and put into the homogenization grinder. Ice-cold homogenization buffer was added 4:1 (4 mL to 1 g krill), with the minimum amount of 0.44 mL buffer. The krill was homogenized

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with 10 slow strokes (900 rpm) where the grinder was surrounded by ice to avoid temperature increase.

The homogenate was poured into pre-cooled LoBind eppendorf vials, kept on ice until all four krill were homogenized, and then centrifuged for 20 minutes at 12 000 g (4 °C). The supernatant, carefully avoiding the fatty layer, was transferred to a new vial and the centrifugation repeated. The second supernatant was aliquoted in three cryovials (Table 3-7) for storage at -80 °C. Note that every time the krill homogenate was analyzed for total protein, AOPP or MDA, the nature of the homogenate made it necessary to mix (using the pipette) before use to regain homogeneity.

Table 3-7 Distribution of krill homogenate. There was a small volume of krill homogenate available. The minimum volume necessary to perform each analysis was aliquoted to TProt (total protein), AOPP and MDA. The drops were transferred with a glass Pasteur pipette.

Krill thorax TProt AOPP MDA

(g) (drops) (drops) (drops)

0.02-0.07 2 5 remaining

0.08-0.17 2 4 remaining

3.5.1 Total Protein Content

The total protein content in the homogenate was measured based on the principle of Bradford [24] and the Sigma protocol “96 Well Plate Assay Protocol” [25], using BSA as calibration standards (Table 3-2). The krill homogenate was thawed on ice, and pre-diluted in PBS buffer (Table 3-8) to fall within the range of the standard curve (0,1428 -1,2376 mg/mL). The pre-diluted krill homogenate was vortexed and

centrifuged (5000 g, 5 minutes). Blank (PBS buffer), calibration standards, control (Liquichek with expected protein content 0.873 mg/mL) and pre-diluted krill homogenate were all added in triplicates (5 μL), then Bradford Reagent (250 μL) was added to all wells using inverted pipetting with a multichannel pipette. The plate was covered with aluminium foil and shaken on the plate reader (300 rpm, 15 minutes) prior to the measurement at 595 nm.

Table 3-8 Total Protein krill homogenate pre-dilution. The krill homogenate was diluted in PBS buffer based on the weight of the thorax, to ensure the absorbance would fall within the range of the standard curve.

Krill thorax Krill homogenate PBS buffer

Dilution

(g) (μL) (μL)

0.02 - 0.07 20 180 1:10

0.08-0.17 20 280 1:15

3.5.2 Pre-Dilution of Krill Homogenate for Total Protein

The optimal pre-dilution of the krill homogenate in PBS for total protein content measurement was tested with dilutions 1:10 (15 µL homogenate and 135 µL PBS), 1:15, 1:20, 1:25 and 1:30 on each of 10 krill homogenates in PBS.

3.5.3 Sedimentation of Krill Homogenate

The robustness of the krill homogenate with respect to total protein content was investigated by how the samples were prepared and stored.

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The Stability of Undiluted Homogenate stored in the Refrigerator

The homogenization process was done with either one centrifugation or two centrifugations. With a single centrifugation the supernatant was frozen directly as the krill homogenate. With two centrifugations the first supernatant transferred to a clean vial and centrifuged a second time, and the second supernatant was frozen as the krill homogenate. The krill homogenates were taken from the -80 C freezer, thawed and total protein content measured with a 1:15 dilution in PBS. The undiluted homogenates were kept in

refrigerator, and reanalyzed two days later diluted 1:15 in PBS.

The Stability of Pre-Diluted Homogenate stored in the Refrigerator

The krill homogenates were taken from the -80 °C freezer, thawed and the total protein content measured in a small portion after 1:20 dilution in PBS. The undiluted homogenates were kept in refrigerator, and another small portion reanalyzed at dilution 1:20 in PBS on four executive days.

Pre-Diluted Homogenate Stability in Refrigerator

The krill homogenates were taken from the -80 °C freezer, thawed and pre-diluted to 1:20 in PBS. The pre-diluted homogenates were kept in refrigerator, vortexed and centrifuged (5000 g, 5 minutes) prior to analysis. The total protein content were analyzed in small aliquots of the diluted homogenate at day 1 (the same day as thawed), day 2, day 3, day 8, day 9, day 10 and day 11.

3.6 Measurement of AOPP

The AOPP content was analyzed as described by Hanasand et al. [26], with some modifications for the krill samples. The samples were added in triplicates to the wells of the UV transparent 96-microtiter plate in two steps (Table 3-9). The first step was to add 190 μL of blank (0.2 M citric acid) and calibration standards (Table 3-3) to the first three columns, and then 40 μL of control and pre-diluted krill

homogenate (Table 3-10) were added to the remaining columns of the microplate. The second step was to add KI (1.16 M, 10 μL) to the blank and the calibration standards, and then protect the plate from further light exposure by gradually covering the plate with aluminium foil while adding citric acid (0.2 M, 160 μL) to the pre-diluted homogenate and controls. After 2 minutes (300 rpm) on the shaker in the plate reader, the wells were briefly inspected for bubbles. The absorbance (340 nm) was measured 5, 10, 15 and 20 minutes after adding KI. The resulting AOPP concentration is reported in μM chloramine-T

equivalents, abbreviated to μM.

Table 3-9 Outline of 96-microtiter plate for AOPP. Columns 1 to 3 were used for 190 μL of calibration standards and blank in triplicates, and 10 μL KI was added in the second step. Columns 4 to 12 were used for 40 μL of control samples and krill homogenates in triplicates, and 160 μL of citric acid (0.2 M) was added in the second step.

1 - 3 4 – 6 7 - 9 10 -12

A Blank x 3 Control x 3 Krill homogenate x 3 Krill homogenate x 3 B Cal 1 x 3 Krill homogenate x 3 Krill homogenate x 3 Krill homogenate x 3 C Cal 2 x 3 Krill homogenate x 3 Krill homogenate x 3 Krill homogenate x 3 D Cal 3 x 3 Krill homogenate x 3 Krill homogenate x 3 Krill homogenate x 3 E Cal 4 x 3 Krill homogenate x 3 Krill homogenate x 3 Krill homogenate x 3 F Cal 5 x 3 Krill homogenate x 3 Krill homogenate x 3 Krill homogenate x 3 G Cal 6 x 3 Krill homogenate x 3 Krill homogenate x 3 Krill homogenate x 3 H Cal 7 x 3 Krill homogenate x 3 Krill homogenate x 3 Krill homogenate x 3

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Table 3-10 AOPP krill homogenate pre-dilution. The krill homogenate was diluted in PBS buffer based on the weight of the thorax, to ensure the absorbance would fall within the range of the standard curve.

Krill thorax (g)

Krill homogenate

(μL) PBS buffer

(μL) Dilution^a

0.02 - 0.07 24 16 1:8.3

0.08 -0.17 15 25 1:13.3

aDilution refers to the final dilution of krill homogenate in the well when 160 μL of citric acid is added.

A large human plasma pool was frozen in small aliquots and used as control. During method development, the krill homogenate was pre-diluted in different solvents to find out if the solvent had any effect on the sample stability and absorbance reading; citric acid (0.2M and 0.5 M), buffer A and PBS buffer. The combination of 40 μL pre-diluted homogenate and 160 μL citric acid was fixed.

3.6.1 Stability of KI and Calibration Standards

Chloramine-T with KI as calibration standard was investigated by looking at how light sensitivity and storage of KI influenced the absorbance over time. The AOPP absorbance readings (340 nm) were performed at short time intervals. Freshly made KI (1.16 M, 10 μL) was compared to one and three weeks old KI (1.16 M, 10 μL) protected from light exposure on sets of calibration standards. The light sensitivity of KI was tested by comparing absorbance of calibrator solutions on plates covered with aluminium foil to the absorbance of the calibrator solution on plates not covered with aluminum foil.

3.6.2 Pre-Dilution of Krill Homogenate for AOPP

The optimal pre-dilution and solvent was investigated for plasma and for krill homogenate. The pre- dilution with PBS and buffer A was done prior to samples added to microplates. The citric acid ‘pre- dilution’ was done by first adding only plasma sample to the well (< 40 μL). The additional volume of citric acid for pre-dilution was added at the same time as the 160 μL of citric acid was added.

 The plasma control was pre-diluted in citric acid (0.2 M), PBS buffer and buffer A respectively, to achieve the final dilution on plate as 1:5, 1:10, 1:13.3 or 1:20, after 40 μL of the pre-diluted plasma had been added 160 μL citric acid (0.2 M). The AOPP content was measured.

 The krill homogenate was pre-diluted in citric acid (0.2 M) and PBS respectively, to achieve the final dilution on plate as to 1:10, 1:13.3 or 1:20 after 40 μL of the pre-diluted krill homogenate had been added 160 μL citric acid (0.2 M). The AOPP content was measured.

3.6.3 Citric Acid Concentration

The pre-diluted krill homogenates (40 μL) were dissolved in citric acid (160 μL) in the AOPP procedure.

The optimal citric acid concentration was investigated by considering the stability of measured AOPP and by visual inspection of the clarity the diluted samples in cuvettes.

 40 μL of pre-diluted krill homogenate in PBS (1:13.3) was added 160 μL of citric acid (0.1, 0.2, 0.5 or 1 M) to achieve a total dilution of 1:15. The AOPP content was measured.

 The pre-diluted krill homogenate in PBS (1:13.3) was diluted with citric acid concentrations (0.1 or 0.2 M). Solutions with different concentrations were visually compared in cuvettes.

Detection of oxidative stress in tissue homogenate from krill exposed to oil

Faculty of Science and Technology

MASTER’S THESIS

Detection of oxidative stress in tissue homogenate from krill exposed to oil

Detection of oxidative stress in tissue homogenate from krill exposed to oil

Master’s thesis in Biological Chemistry

Linda Bærheim Spring 2015

Acknowledgement

Abstract

Table of Contents

Abbreviations

1 Introduction

1.1 Krill

1.2 Produced Water

1.3 Krill Exposure Study

1.4 Purpose of Thesis

2 Theory

2.1 Oxidative Stress

2.2 Biomarkers for Oxidative Stress

2.3 Spectrophotometry

2.4 High Performance Liquid Chromatography – Fluorescence

3 Materials and Methods

3.1 Chemicals and Reagents

3.2 Equipment

3.3 Solutions

3.4 Krill

3.5 Homogenization

3.6 Measurement of AOPP